Unraveling the three‐dimensional morphology and dynamics of the optically evolving polystyrene nanoparticle assembly using dual‐objective lens microscopy

Author(s):  
Abdullah Kamit ◽  
Ching‐Shiang Tseng ◽  
Tetsuhiro Kudo ◽  
Teruki Sugiyama ◽  
Johan Hofkens ◽  
...  
Author(s):  
Kenneth H. Downing

Three-dimensional structures of a number of samples have been determined by electron crystallography. The procedures used in this work include recording images of fairly large areas of a specimen at high tilt angles. There is then a large defocus ramp across the image, and parts of the image are far out of focus. In the regions where the defocus is large, the contrast transfer function (CTF) varies rapidly across the image, especially at high resolution. Not only is the CTF then difficult to determine with sufficient accuracy to correct properly, but the image contrast is reduced by envelope functions which tend toward a low value at high defocus.We have combined computer control of the electron microscope with spot-scan imaging in order to eliminate most of the defocus ramp and its effects in the images of tilted specimens. In recording the spot-scan image, the beam is scanned along rows that are parallel to the tilt axis, so that along each row of spots the focus is constant. Between scan rows, the objective lens current is changed to correct for the difference in specimen height from one scan to the next.


Author(s):  
Robert W. Mackin

This paper presents two advances towards the automated three-dimensional (3-D) analysis of thick and heavily-overlapped regions in cytological preparations such as cervical/vaginal smears. First, a high speed 3-D brightfield microscope has been developed, allowing the acquisition of image data at speeds approaching 30 optical slices per second. Second, algorithms have been developed to detect and segment nuclei in spite of the extremely high image variability and low contrast typical of such regions. The analysis of such regions is inherently a 3-D problem that cannot be solved reliably with conventional 2-D imaging and image analysis methods.High-Speed 3-D imaging of the specimen is accomplished by moving the specimen axially relative to the objective lens of a standard microscope (Zeiss) at a speed of 30 steps per second, where the stepsize is adjustable from 0.2 - 5μm. The specimen is mounted on a computer-controlled, piezoelectric microstage (Burleigh PZS-100, 68/μm displacement). At each step, an optical slice is acquired using a CCD camera (SONY XC-11/71 IP, Dalsa CA-D1-0256, and CA-D2-0512 have been used) connected to a 4-node array processor system based on the Intel i860 chip.


2020 ◽  
Vol 10 (18) ◽  
pp. 6243
Author(s):  
Seohyun Lee ◽  
Hyuno Kim ◽  
Hideo Higuchi

Imaging the three-dimensional movement of small organelles in living cells can provide key information for the dynamics of drug delivery and virus transmission in biomedical disciplines. To stably monitor such intracellular motion using microscope, long depth of field along optical axis and accurate three-dimensional tracking are simultaneously required. In the present work, we suggest an extended dual-focus optics microscopy system by combining a bifocal plane imaging scheme and objective lens oscillation, which enables accurate localization for a long axial range. The proposed system exploits high-resolution functionality by concatenating partial calibration result acquired each axial imaging level, maintaining the practical advantages of ratiometric method.


Micromachines ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 293
Author(s):  
Wenhao Du ◽  
Cheng Fei ◽  
Junliang Liu ◽  
Yongfu Li ◽  
Zhaojun Liu ◽  
...  

Optical projection tomography (OPT) is the direct optical equivalent of X-ray computed tomography (CT). To obtain a larger depth of field, traditional OPT usually decreases the numerical aperture (NA) of the objective lens to decrease the resolution of the image. So, there is a trade-off between sample size and resolution. Commercial microfluidic systems can observe a sample in flow mode. In this paper, an OPT instrument is constructed to observe samples. The OPT instrument is combined with commercial microfluidic systems to obtain a three-dimensional and time (3D + T)/four-dimensional (4D) video of the sample. “Focal plane scanning” is also used to increase the images’ depth of field. A series of two-dimensional (2D) images in different focal planes was observed and compared with images simulated using our program. Our work dynamically monitors 3D OPT images. Commercial microfluidic systems simulate blood flow, which has potential application in blood monitoring and intelligent drug delivery platforms. We design an OPT adaptor to perform OPT on a commercial wide-field inverted microscope (Olympusix81). Images in different focal planes are observed and analyzed. Using a commercial microfluidic system, a video is also acquired to record motion pictures of samples at different flow rates. To our knowledge, this is the first time an OPT setup has been combined with a microfluidic system.


2015 ◽  
Vol 656-657 ◽  
pp. 549-553
Author(s):  
Kyohei Nishimoto ◽  
Kozo Taguchi

Dielectrophoresis (DEP) force will arise when an inhomogeneous AC electric field with sinusoidal wave is applied to microelectrodes. By using DEP, we could distinguish between viable and non-viable cells by their movement through a non-uniform electric field. In this paper, we propose a yeast cell separation system, which utilizes an Au DEP chip and an optical tweezers. The Au DEP chip is planar quadrupole microelectrodes, which were fabricated by Au thin-film and a box cutter. This fabrication method is low cost and simpler than previous existing methods. The tip of the optical tweezers was fabricated by dynamic chemical etching in a mixture of hydrogen fluoride and toluene. The optical tweezers has the feature of high manipulation performance. That does not require objective lens for focusing light because the tip of optical tweezers has conical shape. By using both the Au DEP chip and optical tweezers, we could obtain three-dimensional manipulation of specific cells after viability separation.


Langmuir ◽  
2010 ◽  
Vol 26 (14) ◽  
pp. 11690-11698 ◽  
Author(s):  
Sun Choi ◽  
Stefano Stassi ◽  
Albert P. Pisano ◽  
Tarek I. Zohdi

Author(s):  
Karen F. Han ◽  
Alexander J. Gubbens ◽  
Abraham J. Koster ◽  
Michael B. Braunfeld ◽  
John W. Sedat ◽  
...  

The primary project of our laboratory is the investigation of chromatin structure by three dimensional electron microscope tomography. The goal is to understand how 30nm fibers fold into higher order chromatin structures. Three dimensional tomography involves the reconstruction of an object by combining multiple projection views of the object at different tilt angles. Due to the electronspecimen interaction and the characteristics of lens aberration in the electron microscope, however, the image is not always an accurate representation of the projected object mass density. In this abstract, we analyze the various types of electron-specimen interaction for thick biological specimens up to 0.7 microns thickness.Electron-specimen interactions include single elastic and inelastic, and multiple elastic and inelastic scattering. Of the imaging electrons, the single elastic and the plasmon electrons give rise to image intensities that can be linearly related to the projected object mass density. Multiply scattered elastic electrons contribute to an increase in background intensity. In addition, due to the chromatic aberration of the TEM’s objective lens, multiply scattered inelastic electrons cause a blurring of the image because of an effective broadening of the focus spread.


MRS Bulletin ◽  
2004 ◽  
Vol 29 (3) ◽  
pp. 177-181 ◽  
Author(s):  
Ian K. Robinson ◽  
Jianwei Miao

AbstractX-rays have been widely used in the structural analysis of materials because of their significant penetration ability, at least on the length scale of the granularity of most materials. This allows, in principle, for fully three-dimensional characterization of the bulk properties of a material. One of the main advantages of x-ray diffraction over electron microscopy is that destructive sample preparation to create thin sections is often avoidable. A major disadvantage of x-ray diffraction with respect to electron microscopy is its inability to produce real-space images of the materials under investigation—there are simply no suitable lenses available. There has been significant progress in x-ray microscopy associated with the development of lenses, usually based on zone plates, Kirkpatrick–Baez mirrors, or compound refractive lenses. These technologies are far behind the development of electron optics, particularly for the large magnification ratios needed to attain high resolution. In this article, the authors report progress toward the development of an alternative general approach to imaging, the direct inversion of diffraction patterns by computation methods. By avoiding the use of an objective lens altogether, the technique is free from aberrations that limit the resolution, and it can be highly efficient with respect to radiation damage of the samples. It can take full advantage of the three-dimensional capability that comes from the x-ray penetration. The inversion step employs computational methods based on oversampling to obtain a general solution of the diffraction phase problem.


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